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Abstract

In this study we show that it is possible to successfully combine the benefits of light-sheet microscopy, self-reconstructing Bessel beams and two-photon fluorescence excitation to improve imaging in large, scattering media such as cancer cell clusters. We achieved a nearly two-fold increase in axial image resolution and 5–10 fold increase in contrast relative to linear excitation with Bessel beams. The light-sheet penetration depth could be increased by a factor of 3–5 relative to linear excitation with Gaussian beams. These finding arise from both experiments and computer simulations. In addition, we provide a theoretical description of how these results are composed. We investigated the change of image quality along the propagation direction of the illumination beams both for clusters of spheres and tumor multicellular spheroids. The results reveal that light-sheets generated by pulsed near-infrared Bessel beams and two photon excitation provide the best image resolution, contrast at both a minimum amount of artifacts and signal degradation along the propagation of the beam into the sample.

Fig. 2Simulated fluorescent beam profiles. Simulated fluorescence cross-sections F(x, y) for different types of illumination beams with the same depth of field along the illumination z-axis. A Gaussian beam for one-photon (1st column) and two-photon excitation (2nd column) and a Bessel beam for one-photon (3rd column) and two-photon excitation (4th column). a) Single beam fluorescence intensity F(x, y, 0) and the corresponding line scans F(0, y, 0) on the right. b) Light sheet fluorescence produced by a lateral scan ∫ F(x, y, 0)dx of the three beams in x-direction and line profiles ∫ F(0, y, 0)dx|x=0 through the light-sheets. c) Multiplications of the light-sheets with the detection point-spread function hdet(x, z).

Fig. 3Measured linear and non-linear fluorescence excitation. The fluorescence excited by Bessel beams in a fluorescein solution is shown for linear fluorescence excitation (a) and TPE (b). In the linear case, the Bessel beams ring system is strongly visible. In contrast, in the case of TPE the ring system is efficiently suppressed so that fluorescence is excited almost exclusively by the beams main lobe. This effect is quantified by the line-scans shown in (c), where hardly any changes in the profiles for different distances z are visible.

Fig. 4Measured fluorescence excited by a Bessel beam in a scattering medium. The fluorescence excited by Bessel beams over a distance of 320μm in a slightly fluorescent gel containing fluorescent 0.75μm polystyrene spheres is shown for linear fluorescence excitation (a) and for TPE (b). In the linear case, the Bessel beams ring system illuminates many spheres outside the Bessel beams main lobe. For TPE, fluorescence excitation by the ring system and by scattered light is suppressed so that mainly spheres along a thin line corresponding to the Bessel beams main lobe are visible.

Fig. 5Simulated images of beads embedded in a fluorescing gel illuminated with four different beams. All beams propagate in z-direction (from left to the right). Each image p(x, y0, z) is shown above a cross-section p(x0, y, z) along the detection axis. The positions x0 and y0 are indicated by dashed lines. a) 1p- Gaussian beam. b) 2p- Gaussian beam. c) 1p-Bessel beam. d) 2p- Bessel beam. e) 200nm thin ideal light sheet without scattering. The LUT of the images is auto-scaled so that they express the relation between the amplitude of the artifacts and the images of the spheres. Note that no information on the absolute image contrast can be drawn from these images. f, g) the lateral standard-deviation of the ideal and the ghost image for all four imaging modes, according to Eq. (15) derived in [2]. Qart gives the ratio of the total lateral standard-deviations of the ideal and the ghost images according to Eq. (15).

Fig. 6Measured images of fluorescing spheres for linear and two-photon Bessel beam excitation. Measured images of fluorescing polystyrene spheres (d = 0.75μm) embedded in a gel illuminated with two different Bessel beams, which propagate from left to right. The image projection ∑yp(x, z) along the detection axis y is shown above the projection ∑xp(y, z) along x. a) single photon fluorescence excitation and b) two-photon fluorescence excitation. Magnification for single-photon excitation and two-photon excitation taken from the area marked by a dashed rectangle in b) and e) are shown in c) and f), respectively. The total size of the image to volume is 75μmx40μmx325μm.

Fig. 8Images of a tumor multicellular spheroid for scanned illumination beams. a–c) show slices p(x, y0, z) from the same tumor multicellular spheroid with a diameter of 230 μm. The insets show magnified parts at the back side of the spheroid that are marked by white boxes. The images were acquired y0 = 100μm inside the spheroid. All beams which are a) 1p- Gaussian beam, b) 1p- Bessel beam and c) 2p- Bessel beam have the same depth of field and propagate from left or right. d) shows the intensity decay p(z) along the propagation z-direction for single illumination beams, acquired by multiplication of the images of the static beam at 400 positions with a Gaussian mask (1/e-width of 750nm) and integration along x.

Fig. 92p-Images of a tumor multicellular spheroid with and without confocal line detection. Image slices p(x, y0, z) from the same spheroid illuminated by a Bessel beam and two-photon fluorescence excitation. Image planes were required at y0 = 120μm (top row) and y0 = 40μm (bottom row) inside the spheroid. Fluorescence images were acquired by a scanned illumination beam (a,c) and by confocal line-detection (b,d). e) The signal-to-background Qcon(yi) = HSF(yi)/LSF(yi) generated from four different illumination and detection schemes for various image planes in detection direction yi. Examples for the high- and low-pass filtered images, pHSF(x, y0, z) and pLSF(x, y0, z) that used to generate the contrast parameter Qcon = HSF/LSF are shown in (f).